Although glucocorticoids (GCs) are widely used to treat allergic and autoimmune diseases, their application is accompanied by severe side effects, including detrimental effects on the skeleton being one of the major complications (James et al., 2007). GC-induced reduction of bone mass by GCs is believed to involve systemic effects and/or direct effects on bone cells (Canalis et al., 2007) leading to induction of apoptosis in osteoblasts and osteocytes (Weinstein et al., 1998) and/or suppression of their differentiation. Whereas low doses of GCs stimulate (Shalhoub et al., 1992), high doses inhibit osteoblast differentiation, the latter being related to the inhibition of postconfluential proliferation (Smith et al., 2000). Suppression of osteoblast function could be a consequence of decreased expression of genes involved in bone formation, such as collagen type 1 (Col1a1) and Runt-related transcription factor 2 (Runx2), or could be due to antagonizing the bone morphogenetic protein (BMP) pathway and the Wnt signaling pathways (Canalis et al., 2007). Besides osteoblasts, modulation of osteoclast activity also appears to contribute to GC-induced osteoporosis (GIO). GCs induce the synthesis of receptor activator for nuclear factor-κB ligand (RANKL), an essential stimulator of osteoclastogenesis (Hofbauer et al., 1999). In addition, they prolong the longevity of osteoclasts in vivo, but decrease their bone degrading activity (Jia et al., 2006; Kim et al., 2006).
The majority of GC effects are mediated via the glucocorticoid receptor (GR), a widely expressed member of the nuclear receptor superfamily. Following hormone binding, the GR alters gene expression via several modes of action, including its binding as dimers to GC-responsive elements (GRE) present in the promoter of hormone-responsive genes and interaction of the monomeric receptor with DNA-bound transcription factors such as NF-KB, AP-1, IRF-3 or STAT5 (Kassel and Herrlich, 2007). Currently, suppression of these transcription factors is believed to underlie in part the anti-inflammatory effects of GCs, while dimerization of the GR is hypothesized to contribute to many of the side effects.
This view is supported by the finding that interaction with pro-inflammatory transcription factors is preserved in GRdim mice carrying a dimerization-deficient GR, while induction of GRE-dependent transcription is abolished (Reichardt et al., 1998; Reichardt et al., 2001). However, there is a requirement for GR dimerization to achieve full suppression of inflammation in contact allergy, septic shock and arthritis, as GC treatment is therapeutically inefficient in GRdim mice (Tuckermann et al., 2007) [Baschant et al. 2011, Kleiman et al. 2011].
The currently accepted paradigm regarding glucocorticoid therapy and GR activity is that in the absence of GC treatment, no GR-mediated gene regulation takes place, whereby AP-1 and NF-KB activity (amongst other transcription factors) leads to a profile of gene expression that induces an inflammatory response. During GC therapy GR function is altered, leading to GR-dimerization dependent gene activation, suggested to be involved in side effects, and GR-monomer dependent transrepression. In the latter case GR-dependent transcription factors such as NF-KB and AP-1 are repressed, therefore repressing the gene expression profile downstream of NF-KB and AP-1 that is associated with inflammation. It is thought that through the repression of NF-KB and AP-1 the anti-inflammatory effect of GC therapy is achieved. Both NF-KB and AP-1 have until now always been co-inhibited via GR during GC treatment. There have existed until now no compounds known to modulate NF-KB and AP-1 activities independently. Despite this effective anti-inflammatory treatment, the significant side effect of GC-induced osteoporosis (GIO) remains and until now remains poorly understood.
The publications and other materials, including patents, used herein to illustrate the invention and, in particular, to provide additional details respecting the practice are incorporated herein by reference in their entirety.
The present invention demonstrates that GR-mediated repression of AP-1 activity during GC therapy plays an important role in GIO. One of the important functions of AP-1 in preventing GIO is driving LIF expression, as shown in the examples below.
Leukaemia inhibitory factor (LIF) is a secreted polyfunctional cytokine which elicits a diversity of biological effects on many cell types. LIF expression has been detected in a variety of cell lines and primary tissues, including primordial germ cells, neurons, embryonic stem cells, adipocytes, hepatocytes and osteoblasts (reviewed Metcalf, 1992 (Growth Factors 7, 169-173); Heath, 1992 (Nature 359, 17)). The biological activities attributed to LIF are widespread. LIF stimulates T lymphocytes and monocytes, brain glial cells, liver fibroblasts, bone marrow stromal cells, thymic epithelial cells and uterine endometrial gland cells just prior to blastocyst implantation. However, until now there has been no mention of LIF in combination with a role in GIO.
The invention relates to agents and/or methods that selectively inhibit NF-KB through the GR, but have no inhibitory effect on AP-1 activity, or maintain or restore either AP-1 activity or one or more of the effects of AP1 activity, such as LIF expression, by maintaining LIF expression or by treatment with LIF.
These agents and/or methods therefore exhibit anti-inflammatory effects due to their NF-KB inhibitory effects but do not lead to GIO due to either an absence of AP-1 inhibitory activity or a restoration of AP-1 activity or maintenance or restoration of AP-1 mediated effects, by maintaining LIF expression or treatment with LIF.
Until now it was unknown that AP-1 or LIF played a role in GIO. It was also unknown that GR-transrepression could be influenced using particular agents to de-couple GR-mediated repression of NF-KB from AP-1 repression, therefore providing anti-inflammatory properties that do not simultaneously cause GIO.
Documents WO 2009/092796 A1 (compare U.S. Pat. No. 8,039,501) and WO 2003/073991 A2 (U.S. Pat. No. 7,655,420) mention the potential avoidance of GIO, although explain the avoidance of GC side effects by reciting the importance of compounds that do not lead to GR-transactivation. Neither disclosure reveals the inventive concept of the present invention; that NF-KB and AP-1 (or the down-stream effects of AP-1) should be differentially regulated in maintaining inflammatory properties that do not simultaneously cause GIO.
Further substances have been proposed in the prior art that are useful as anti-inflammatory compounds (WO 2009/058944 A2 (U.S. Pat. No. 8,309,730), WO 2006/076509 A1 (U.S. Pat. No. 7,317,024)), although such compounds do not exhibit the inventive properties of the present invention. Although AP-1 and LIF activity have been associated with bone-density, there has until now been no disclosure or suggestion that AP-1 or LIF play an important role in GC-induced osteoporosis.
Other approaches towards enhancing bone density, such as dexamethasone treatment, have no stimulatory effect on AP-1 at pharmacologically relevant doses (WO 2003/073991 A2 (U.S. Pat. No. 7,655,420), Kurahashi et al., 2005). As also described above, a stimulating effect for GCs on bone density has been previously described, however, at pharmacologically relevant doses GCs lead to the opposite effect, so that bone density is in fact reduced.
Compound A and its effect as an alternative to known glucocorticoids has been disclosed in the prior art (U.S. Pat. No. 7,053,120 B2, van Loo et al., 2010, Gossye et al., 2010, Wuest et al., 2009, Gossye et al., 2009, Zhang et al., 2009, Haegeman et al., 2006, De Bosscher et al., 2005). However, until the present time it was unknown that Compound A exhibits the inventive properties as described herein.
Other compounds have also been disclosed that could be of use in the treatment of GIO (WO 2004/106296 A2 (U.S. Pat. No. 7,459,460), WO 2004/106295 A2 (U.S. Pat. No. 7,265,145), WO 2005/077925 A1 (U.S. Pat. No. 7,820,702)). All such disclosures make however no mention of an effect on either AP-1 or GR.